Trophoblasts play an essential role in communication between the fetus and the mother. Failure to accurately establish the trophoblast lineage results in developmental arrest prior to the blastocyst stage, implantation failure, early miscarriage, and placental abnormalities. Recently, we made the first discovery of a novel DNA methylation on N6-adenine (N6-mA) in mammalian genomes (Wu et al., Nature, 2016; Xie et al., Cell, 2018). Our most recent preliminary studies have shown that N6-mA is predominantly present in the trophectoderm/trophoblast lineage of early embryos, and is conserved in the mouse and bovine and probably across all mammals. Alkbh1 (N6-mA demethylase)-deficient in mice die in utero due to loss of imprinted control and severe placental development defects in post-implantation embryos. Interestingly, ALKBH1 deficient bovine embryos have impaired trophoblast stem cell emergence and blastocyst formation. In addition, N6-mA abundance is remarkably high in trophoblast cultures differentiated from human pluripotent stem cells, providing an excellent model for studying N6-mA in human placentation. These results indicated that this novel epigenetic mark has both conserved and unique roles in trophoblast development in different eutherians. However, the molecular mechanism underlying N6-mA function, and its role in trophectoderm and trophoblast development remain elusive. The overall hypothesis is that N6-mA represents a novel epigenetic mark that regulates gene expression network directing determination of trophoblast cell fate. The overall objective of the proposed studies is to conduct a systematic, comparative study on trophoblast from mice and bovine embryos, as well as human trophoblast cell culture models, focusing on the role of N6-mA in trophoblast formation, gene expression and epigenetic reprogramming. Three independent and yet intellectually-related specific aims are proposed. Aim 1 will characterize N6-mA function in trophoblast lineage differentiation and imprinted gene control with a trophoblast-specific conditional knockout mouse model. Aim 2 will investigate the role of N6-mA in trophoblast stem cell emergence and blastocyst formation in a bovine embryo model. Aim 3 will utilize human stem cell-derived trophoblast differentiation models to establish N6-mA functions in human placental trophoblast formation. This project leverages the expertise of the Jiang laboratory in embryo/trophoblast development and the expertise of the Xiao laboratory in epigenetic regulation to address fundamental questions in trophoblast lineage development. This study is innovative because it a) provides insight into the role of a novel DNA methylation mark in trophoblast lineage formation, b) sheds new light on epigenetic regulation of imprinted genes, a longstanding issue in placental development, and c) establishes novel, comparative embryo and cell culture models to understand the conserved and unique epigenetic regulation of trophoblast cell fate in eutherians. The proposed project is significant because it will improve our understanding of the molecular basis of early embryonic loss and clinical reproductive disorders that are associated with abnormal placentation.